Incorporation of membrane proteins into large single bilayer vesicles. Application to rhodopsin.

A general procedure to incorporate membrane proteins in a native state into large single bilayer vesicles is described. The results obtained with rhodopsin from vertebrate and invertebrate retinas are presented. The technique involves: (a) the direct transfer of rhodopsin-lipid complexes from native membranes into ether or pentane, and (b) the sonication of the complex in apolar solvent with aqueous buffer followed by solvent evaporation under reduced pressure. The spectral properties of rhodopsin in the large vesicles are similar to those of rhodopsin in photoreceptors; furthermore, bleached bovine rhodopsin is chemically regenerable with 9-cis retinal. These results establish the presence of photochemically functional rhodopsin in the large vesicles. Freeze-fracture replicas of the vesicles reveal that both internal and external leaflets contain numerous particles approximately 80 A in diameter, indicating that rhodopsin is symmetrically distributed within the bilayer. More than 75% of the membrane area is incorporated into vesicles larger than 0.5 micron and approximately 40% into vesicles larger than 1 micron

Mitochondria, glutamate neurotoxicity and the death cascade

AbstractThis review focuses on two questions: the role of mitochondria in excitotoxic neuronal death and the connection of mitochondria with the apoptotic death cascade. The goal is to highlight the regulatory role of mitochondrial channels on the mitochondrial membrane potential, Δψ, and their involvement in determining neuronal survival or death. A hypothesis is developed centered on the notion that protein–protein interactions between members of the Bcl-2 family of death suppressor and promoter proteins lead to the selective elimination of depolarizing currents that, in turn, collapse Δψ and set in motion the irreversible pathway of cell death. The model considers the remarkable propensity of Bcl-2 family proteins to dimerize or oligomerize and thereby restrict the localization of partner molecules to mitochondrial membrane contact sites. The fundamental principle invoked here is that through a concerted set of protein–protein interactions, information is exchanged by specific heterodimers, one of the partners acting as a toxic protein and the second as its antidote. The review concludes with the elaboration of a speculative model about cellular mechanisms for the prevention of cell destruction as triggered by extracellular signals which may be conserved in its molecular design from bacteria to eukaryotes

Design, synthesis and functional characterization of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor

AbstractNicotinic cholinergic receptors are membrane proteins composed of five subunits organized around a central aqueous pore. A pentameric channel protein, T5M2δ, that emulates the presumed pore-forming structure of this receptor was generated by assembling five helix-forming peptide modules at the lysine ϵ-amino groups of the 11-residue template [K∗AK∗KK∗PGK∗EK∗G], where ∗ indicates attachment sites. Helical modules represent the sequence of the M2 segment of the Torpedo californica acetylcholine receptor (AChR) δ subunit; M2 segments are considered involved in pore-lining. Purified T5M2δ migrates in SDS-PAGE with an apparent Mr~14,000, concordant with a protein of 126 residues. T5M2δ forms cation-selective channels when reconstituted in planar lipid bilayers. The single channel conductance in symmetric 0.5 M K.C1 is 40 pS. This value approximates the 45 pS single channel conductance characteristic of authentic purified Torpedo AChR, recorded under otherwise identical conditions. These results, together with conformational energy calculations, support the notion that a bundle of five amphipathic a-helices is a plausible structural motif underlying the inner bundle that forms the pore of the pentameric AChR channel

Mètodes diagnòstics instrumentals de la circulació arterial de les extremitats inferiors

Treball Final de Grau de Podología, Escola Universitaria d'Infermeria, Universitat de Barcelona, curs: 2013-2014, Tutor: Carolina Padrós SánchezDegut a l’elevada prevalença de la malaltia arterial perifèrica (MAP), que amb freqüència és asimptomàtica, i les seves greus repercussions de pronòstic, es recomana fer un cribatge a l'hora del diagnòstic dels pacients amb alt risc de patir-la. Tant l’anamnesis com la exploració física tenen un valor per al diagnòstic de la malaltia limitat, ja que encara que siguin molt específics, la seva sensibilitat és molt baixa. Existeixen múltiples proves diagnòstiques per valorar la presència i la gravetat de la malaltia vascular perifèrica, entre les quals cal destacar l’índex tormell-braç, determinacions de pressions segmentàries, estudis d’ecografia doppler i angiografia mitjançant ressonància magnètica, tomografia computaritzada o de sustracció digital. A l’actualitat es considera que l’índex tormell-braç és el mètode no invasiu que presenta millor rendiment de diagnòstic, ja que es tracta d’una prova fàcil de realitzar i amb una elevada sensibilitat (superior al 90%)

Event-based media monitoring methodology for Human Rights Watch

Executive Summary This report, prepared by a team of researchers from the University of Minnesota for Human Rights Watch (HRW), investigates the use of event-based media monitoring (EMM) to review its application, identify its strengths and weaknesses, and offer suggestions on how HRW can better utilize EMM in its own work. Media monitoring systems include both human-operated (manual) and automated systems, both of which we review throughout the report. The process begins with the selection of news sources, proceeds to the development of a coding manual (for manual searches) or “dictionary” (for automated searches), continues with gathering data, and concludes with the coding of news stories. EMM enables the near real-time tracking of events reported by the media, allowing researchers to get a sense of the scope of and trends in an event, but there are limits to what EMM can accomplish on its own. The media will only cover a portion of a given event, so information will always be missing from EMM data. EMM also introduces research biases of various kinds; mitigating these biases requires careful selection of media sources and clearly defined coding manuals or dictionaries. In manual EMM, coding the gathered data requires human researchers to apply codebook rules in order to collect consistent data from each story they read. In automated EMM, computers apply the dictionary directly to the news stories, automatically picking up the desired information. There are trade-offs in each system. Automated EMM can code stories far more quickly, but the software may incorrectly code stories, requiring manual corrections. Conversely, manual EMM allows for a more nuanced analysis, but the investment of time and effort may diminish the tool’s utility. We believe that both manual and automated EMM, when deployed correctly, can effectively support human rights research and advocacy

Functional reassembly of membrane proteins in planar lipid bilayers

Recent progress in membrane biology has brought us to a stage where it is possible to associate complex biological processes to identifiable membrane proteins. Technical advances in the biochemical characterization and purification of membrane proteins have contributed a wealth of structural information. The reconstitution approach has proved to be valuable in our efforts to understand the molecular mechanisms of membrane transport and energy transductio

Rage against the regime: Policy responses to international investment arbitration

University of Minnesota Ph.D. dissertation. August 2019. Major: Political Science. Advisors: Ronald Krebs, John Freeman. 1 computer file (PDF); xv, 454 pages.Common in political discourse and academic literature is the notion that the international investment regime is experiencing backlash. At the center of this backlash is the belief that international investment treaties unduly restrain states’ ability to regulate in the public interest, most notably by allowing foreign investors to file international arbitration claims directly against governments for a variety of regulatory acts. The rise of investment arbitration--also know as investor-state dispute settlement (ISDS)--has provoked a regime-wide reassertion of states' autonomy to regulate foreign investment under investment treaties. However, states have embarked on this process in different ways and to different extents. While some have become more cautious of how much legal autonomy they sacrifice in the future, others have partially or completely recovered autonomy lost to previous treaties. My dissertation explains why states pursue different policies in the aftermath of ISDS. I argue that negative ISDS experiences are filtered through ideational lenses that incline policy-makers to reclaim more or less legal autonomy. Yet policy-makers face constraints and opportunities when acting on these emerging preferences. Thus, I also argue that policy outcomes depend on the combination of a domestic and external variables. Most scholarly attention has been placed on the political behavior of economic actors, either domestic firms or foreign investors. However, ISDS disputes also affect a broad and diverse ensemble of local and transnational civil society groups. These actors have competing interests regarding continuity and change in investment treaty policies. Thus, I examine the conditions under which their mobilization can enhance or hinder policy-makers' ability to implement their desired policies. I test the expectations derived from this argument using a mixed methods research design that combines quantitative statistical analysis and qualitative case studies. Through regression analysis of an original measure of international legal autonomy, I show that after ISDS claims hit, states are less willing to sacrifice their legal autonomy and in some cases start to recover it. Further analysis of three original datasets of treaty signature, treaty content and treaty termination shows these actions are not equally likely across states. Through within- and cross-case comparisons of investment treaty policy-making in the United States, Ecuador and India, I show how alternative combinations of the explanatory variables make a given policy reaction to ISDS more likely. Whether states continue to endorse strong treaty protections is a pressing question, given the recent rise in the number of governments elected on nationalist platforms. There are also normative stakes in the answers to these research questions. Policy variation does not simply revolve around technical legal disagreements; it reflects fundamental disagreements about the limits of state authority in a globalized economy